Infections of Vibrio cholerae can result in death within a few hours, while enterotoxigenic Escherichia coli (ETEC) is in the third world a major cause of infant diarrhea and death due to dehydration. Key virulence factors of V. cholerae and ETEC are, respectively, cholera toxin (CT) and Heat-labile Enterotoxin (LT), two closely related and sophisticated hetero-hexameric AB5 toxins. Our investigations aim to answer a series of crucial and unresolved questions concerning the mechanism of toxin secretion across the outer membrane of V. cholerae and ETEC. The proposed research uses crystallographic approaches, complemented by electron microscopy and small angle X-ray scattering studies, to unravel the architecture and functioning of the fascinating bacterial Type 2 Secretion System (T2SS) at the atomic level. The T2SS is responsible for secretion of LT and CT, and a host of other proteins, from the periplasm into the extracellular milieu. A remarkable property of the T2SS is its capability to translocate proteins in folded form across the outer membrane. Determining the three-dimensional structure of the large T2SS is a formidable challenge since this system contains multiple copies of at least 11 different proteins and spans two membrane bilayers. The T2SS occurs in numerous pathogenic bacteria attacking humans, animals and plants. Understanding the T2SS is of extra importance since it is related to the Type 4 Pilin Biogenesis System, which also is occurring in many pathogens and is responsible for a diversity of processes, like protein secretion, protein import and DNA uptake. Given the sophistication of the bacterial toxins and the T2SS secretion system, and their wide-spread occurrence in harmful bacteria, our studies will substantially increase the fundamental scientific knowledge of bacterial pathogenesis. At the same time, our structural data will provide a new basis for developing future therapeutic interventions against bacterial infections. PUBLIC HEALTH RELEVANCE: Many pathogenic bacteria secrete toxins to cause human cells to malfunction, often with disastrous consequences for the human host. The proposed studies will show in atomic detail how cholera toxin and related toxins cross the outer bacterial membrane using a sophisticated and complex two-membrane spanning multi-protein machinery. This knowledge may lead to the development of new anti-bacterials.